Henley, Beverley and Cohen, Bruce N. and Kim, Charlene H. and Gold, Heather D. and Srinivasan, Rahul and McKinney, Sheri and Deshpande, Purnima and Lester, Henry A. (2017) Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression. Journal of Visualized Experiments, 120 (e54981). ISSN 1940-087X

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Abstract

In Parkinson's Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated "tagmentation" to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

Item Type:	Article
Keywords:	Neuroscience; Issue 120; single-cell; RNA-sequencing; cell harvesting; midbrain cultures; dopaminergic neurons; Parkinson's disease;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	11561
Identification Number:	https://doi.org/10.3791/54981
Depositing User:	Beverley Henley
Date Deposited:	01 Nov 2019 16:31
Journal or Publication Title:	Journal of Visualized Experiments
Publisher:	JOVE
Refereed:	Yes
URI:	Publisher
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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